Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein
Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein
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DOI：
10.1124/jpet.109.158782
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发表时间：
2009-11-01
影响因子：
3.5
通讯作者：
Hollenberg, Paul F.
中科院分区：
文献类型：
--
作者：
Lin, Hsia-lien;Zhang, Haoming;Hollenberg, Paul F.
研究了叔丁基苯乙炔（BPA）和叔丁基1-甲基-2-丙炔基醚（BMP）对细胞色素CYP2B1 [野生型（WT）]及其Thr205对Ala突变体（T205A）的失活机理。BPA对WT的失活表现出k（inact）/K-I值为1343 min（-1）mM（-1），分配比为1。BMP对WT的失活表现出33 min（-1）mM（-1）的k（inact）/K-I值和10的分配比。WT的液相色谱/串联质谱分析（LC/MS/MS）显示：1）BPA灭活导致形成蛋白质加合物，其质量增加相当于BPA加一个氧原子的质量，2）BMP灭活导致形成多种血红素加合物，其质量增加均相当于BMP加一个氧原子。LC/MS/MS分析表明，通过GSH与BMP或BPA的乙炔基部分的反应形成谷胱甘肽（GSH）缀合物，其中氧被添加到内部或末端碳。对于T205A的BPA和BMP的失活，k（inact）/K-I值分别被抑制了100倍和4倍，分配比分别增加了9倍和3.5倍。在T205 A被BMP灭活后，仅检测到一种主要的血红素加合物。这些结果表明，F-螺旋中的Thr205在BPA和BMP对CYP2B1的基于机制的失活效率中起重要作用。同源性建模和底物对接的研究，以方便解释的实验结果。
The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2- propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K-I value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K-I value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K-I values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.